Isolation and Characterization of Two Endogenous Phages of Rhodobacter sphaeroides

Abstract

Bacterial strains and bacteriophages. Strains of Rhodobacter sphaeroides were obtained from the German Collection of Micro-organisms (DSM), from W. R. Sistrom, University of Oregon, Ore., USA, or were isolates of this laboratory. The R. sphaeroides strains DSM 159-2 (pink) and DSM 159-1 1 (greenish) were colour variants of strain DSM 159 (red-brown). They were detected among colonies of strain DSM 159 plated on YP-medium (see below). The strains are listed in Table 1. The temperate R. sphaeroides phage 4RsG 1 is an isolate of this laboratory (Duchrow et al., 1985).Media and growth conditions. R. sphaeroides is a facultatively photosynthetic bacterium capable of growing anaerobically in the light (phototrophic) or aerobically (chemotrophic). Strains were grown at 30 "C in 20 mM-L( +)-tartrate-mineral medium (TM-medium; Rode & Gifhorn, 1983) or in rich medium (YP-medium) consisting of 0.3% yeast extract (Merck) and 0.3% trypticase peptone (Beckton Dickinson) adjusted with NaOH to pH 7.0. Stocks were maintained in liquid cultures which were incubated in completely filled screw-cap bottles (100 ml) anaerobically in the light at approximately 3000 lx and then stored at 4 "C. Bacteria on agar plates were incubated aerobically or phototrophically in Gas Pak anaerobic jars in front of a 100 W lamp.For phage growth TM-medium was supplemented with 2.5 rn~-CaCl, and YP-medium with 0.05 g CaCl, I -l , and then termed TMC-and YPC-medium respectively.Isolation and purification of phages. Phages 4RsA and 4RsD were isolated from a stock of the temperate R. sphaeroides phage 4RsG 1 (Duchrow et al., 1985) by plating on R. sphaeroides strain DSM 159-2 using the standard overlay technique and repeated single plaque isolation (Adams, 1959).Buflers. These were TE-buffer, 10 mM-Tris/HCl (pH 8*0), 1 mM-EDTA; TS-buffer, 20 mM-Tris/HCl (pH 7.0), 50 mM-NaC1; SSC-buffer, 15 mM-sodium citrate (pH 7.0), 150 mM-NaC1.Phage stocks. These were prepared by infecting a chemotrophic mid-exponential phase culture of R. sphaeroides DSM 159-2 with a filter-sterilized cleared phage lysate (m.0.i. = 0.1) and then incubating for another 48 h. Subsequently, cells and cell debris were sedimented for 10 min at lOOOOg and filter-sterilized. This procedure routinely provided stocks containing lo9 plaque-forming units (p.f.u.) per ml. Chromosomal DNA and RNA from lysed cells were removed from the stocks by incubation in the presence of DNAase (10 pg ml-I) and RNAase 50 pg ml-I) at 37 "C for 3 h. Phage particles were precipitated by addition of 6% (w/v) PEG 6000 and 0.5 M-NaCl (final concentration) and keeping the suspension at 4 "C for another 14 h. The phages were collected by centrifugation as above, resuspended in 1 ml TE-buffer and extensively dialysed against the same buffer at 4 "C.Electron microscopy. Phage particles were spread onto grids with carbon-coated Formvar support films, negatively stained with 4% (w/v) uranyl acetate (Valentine et al., 1968) and examined with a Philips EM 301 electron microscope.Preparation ofphage and bacterial DNA. Phage DN...